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DISSERFOR TH
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UNIVERSITY OF TUSCIA-VITERBO
PhD in Environmental Science XXIV Cycle – SDS BIO/19 MICROBIOLOGY
New Surface Active Compounds from bacterial strains: production, characterization and potential
application in environmental remediation
Dissertation submitted by Tiziana Beltrani
Tutor: Prof. Maurizio Petruccioli
Co-tutor: Dott. Carlo Cremisini
PhD Coordinator: Prof. Maurizio Petruccioli
TABLE OF CONTENTS
CHAPTER 1 INTRODUCTION 1
I
5 1.1 SUMMARY OF THE THESIS
CHAPTER 2 SCREENING OF BACTERIAL STRAINS ABLE TO PRODUCE SURFACE ACTIVE COMPOUNDS 2.1 INTRODUCTION 8
2.1.1 Microbial surfactants 8
2.1.2 Biosurfactants classification and their microbial origin 9
2.1.2.1 Low and high molecular weight biosurfactants 9
2.1.3 Factors affecting biosurfactant production 11
2.1.4 Towards commercial production of microbial surfactants 13
2.1.5 Functional properties of Surface Active Compounds 14
2.1.6 Screening methods for detection of biosurfactant and
bioemulsifier producers 15
2.1.6.1 Oil Spreading Assay 16
2.1.6.2 Emulsification Assay 16
2.2 EXPERIMENTAL PROCEDURES 16
2.2.1 Bacterial strains 16
2.2.2 Control strains 17
2.2.3 Growth conditions and culture preparation 18
2.2.4 Screening for SACs production 19
2.2.4.1 Oil Spreading Test (OST) 19
2.2.4.2 Emulsification Activity (EA) 19
2.2.5 Pre-optimization of culture conditions 20
II
2.3 RESULTS AND DISCUSSION 20
2.3.1 Production of surface active compounds 20
2.3.2 Pre-optimization of culture medium 22
CHAPTER 3 RECOVERY, PURIFICATION AND PHYSICO-CHEMICAL CHARACTERIZATION OF THE BIOEMULSIFIER PRODUCTION BY Pedobacter sp. MCC-Z 3.1 INTRODUCTION 26
3.1.1 Recovery and purification of SACs 26
3.1.2 Characteristics and properties of SACs 28
3.2 EXPERIMENTAL PROCEDURES 30
3.2.1 Recovery of the extracellular crude bioemulsifier 30
3.2.2 Purification of crude bioemulsifier and emulsifying activity 30
3.2.3 Physico-chemical characterization of Pdb-Z 30
3.2.3.1 Surface tension measurements 30
3.2.3.2 Emulsifying activity with various hydrophobic substrates 31
3.2.3.3 Stability study: temperature, pH and ionic strength tolerance 31
3.3 RESULTS AND DISCUSSION 32
3.3.1 Purification and emulsifying activity 32
3.3.2 Surface properties of Pdb-Z :surface tension and CMC value 32
3.3.3 Emulsifying properties 33
3.3.4 Stability study of Pdb-Z: temperature, pH and ionic strength
tolerance 36
CHAPTER 4 CHEMICAL CHARACTERIZATION OF Pdb-Z
4.1 INTRODUCTION 40
4.1.1 Microbial surfactants and their structure 40
4.2 EXPERIMENTAL PROCEDURES 42
4.2.1 Chemical composition: Proteins, lipids and carbohydrates 42
4.2.2 Fourier Transform Infrared spectroscopy 43
4.2.3 1HNMR spectroscopy and hydrodynamic study of Pdb-Z 43
4.3 RESULTS AND DISCUSSION 44
4.3.1 Chemical composition of Pdb-Z 44
III
4.3.2 Fourier Transform Infrared and 1HNMR spectroscopy 46
4.3.3 Hydrodynamic behaviour 48
CHAPTER 5 FEASIBILITY STUDIES FOR APPLICATION OF Pdb-Z IN SOIL WASHING REMEDIATION 5.1 INTRODUCTION 52
activity and reduction in viscosity of crude oil, make their use feasible for many
application purposes [34].
2.1.2 Biosurfactants classifications and their microbial origin
Biosurfactants are amphiphilic compounds produced on living surfaces, mostly
microbial cell surfaces, or excreted extracellularly and contain hydrophobic and
hydrophilic moieties. These compounds can be classified as low and high molecular
mass bioemulsifiers [5]. The former lower surface and interfacial tensions, whereas the
second are more effective at stabilizing oil in water emulsions.
2.1.2.1 Low and high molecular weight biosurfactants
The low molecular mass bioemulsifiers are generally glycolipids, such as treahalose
lipids, sophorolipids and rhamnolipids, or lipopeptides like surfactin (Table 2.1). The
most commonly known glicolipid bioemulsifiers, rhamnolipids, trehalolipids and
sophorolipids, are disaccharides that are carbohydrates in combination with long-chain
aliphatic acids or hydroxyaliphatic acids. A large number of microorganisms produce
glycolipids.
For example, certain species of Pseudomonas are known to produce large amounts of a
glycolipid, called rhamnolipids, [35], Rhodococcus erythropolis produces trehalose
lipids when the bacterium is grown on n-alkanes [36, 37], while, different species of
yeast Torulopis produce extracellular sophorolipids [38,39].
Several bacteria produce large quantities of cyclic liopeptides. Bacillus subtilis produces
a cyclic lipopeptide, called surfactin, one of the most effective biosurfactants [40, 41]
while, Bacillus brevis produces the cyclosymmetric decapeptide antibiotic gramicidin S
that forms a stable coordination complex [42].
Phospholipids biosurfactants have been synthesized by various researchers using several
bacterial and yeast during growth on n-alkanes. For example, Acinetobacter sp.
9
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
10
produces extracellular membrane vesicles able to form microemulsions of alkanes in
water [43].
A large number of bacterial species from different genera produce exocellular
polymeric surfactants composed of polysaccharides, protein, lipopolysaccharides,
lipoproteins or complex mixtures of high molecular mass. The most thoroughly studied
polymeric biosurfactants are Emulsan, liposan, mannoprotein, and other polysaccharide-
protein complexes (Table 2.1).
Rosenberg and Ron [44] studied the production of a potent polyanionic amphipatic
hetereopolysacharide bioemulsifier by different species of Acinetobacter. Acinetobacter
calcoaceticus RAG-1 produces Emulsan one of the most powerful emulsion stabilizers
known today [6]. Cirigliano and Carman [45] reported the production of Liposan by
Candida lipolytica; this is an extracellular water-soluble emulsifier composed of 83%
carbohydrate and 17% protein. Acinetobacter radioresistens produces Alasan, a
complex of an anionic polysaccharide and protein with a molecular mass of
approximately 1MDa [8].
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
Table 2.1: Major types of biosurfactant produced by microorganisms.
Biosurfactant Producing microorganisms
Reference
Low Molecular mass Glycolipids
Trehalose lipids Rhamnolipids Sophorolipids
Pseudomonas spp. R. erythropolis Arthrobacter sp. Mycobacterium sp. P.aeruginosa T. bombicola (yeast) C. borgoriensis (yeast)
Parra et al.1989 Ristau and Wagner 1983; Kim et al.1990; Li et al 1984 Li et al. 1984 Cooper et al.1989 Rendell et al.1990; Sim et al.1997 Inoue and Itoh 1982; Davila 1997 Cutler and light 1979
Aminoacid-lipids Lipopeptides and lipoprotein
Viscosin Surfactin Gramicidin S Peptide-lipid Serrawettin Polimyxins
P. fluorescens B. subtilis B. brevis B. licheniformis S. marcescens B. polymyxa
Neu and Poralla 1990 Arima et al.1968; wei and Chu 1998 Katz and Demain 1977 Horowitz and Griffen 1991 Matsuyama et al.1991 Suzuki et al. 1965
Fatty acids and phospholipids
Corynomycolic acid Phospholipids
N. erythropolis T. thiooxidans Acinetobacter spp.
MacDonald et al. 1981 Beebe and Umbreit 1971 Kaeppeli and Finnerty 1980
A. calcoaceticus RAG-1 C. lipolytica A. radioreresistens KA53 P. fluorescens C .tropicalis A. calcoaceticus A2 C. utilis A. calcoaceticus BD413 H. eurihalinia B. stearothermophilus
Rosenberg et al. 1979 Cirigliano and Carmen 1984 Navon-Venezia et al. 1985 Persson et al. 1988 Kaeppeli et al. 1984 Rosenberg 1993 Shephered et al. 1995 Kaplan and Rosenberg 1982 Calvo et al. 1998 Gunjar et al. 1995
2.1.3 Factors affecting biosurfactant production
During recently years, there have been numerous reviews that have demonstrated that
the composition and surface properties of the biosurfactant depend not only on the
producer strains, but also on the culture conditions. Thus, the nature of the carbon and
nitrogen sources the related C:N ratio, nutritional limitations, chemical and physical
parameters such as, temperature, aeration rate, divalent cations and pH, influence not
only the amount of biosurfactant produced but also the type of polymer produced [46]
11
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
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The quality and quantity of biosurfactant production is dependent on the nature of the
carbon source [47]. Desai and Banat [29] reported that diesel, crude oil, glucose and
sucrose are good substrates for biosurfactant production. Furthermore, there are some
microorganisms that produce biosurfactants only by using a hydrophobic carbon source,
hydrocarbon or vegetable oil, while others use only carbohydrates in combination or
individually [48].
Different nitrogen compounds have been used for the production of biosurfactants such
as urea, peptone, yeast extract, ammonium sulphate, ammonium nitrate, malt extract and
meat extract. Syldatk et al. [49] reported that nitrogen limitation causes overproduction
of biosurfactant. Ammonium salts and urea are preferred nitrogen sources for
biosurfactant production by Arthrobacter paraffineus, whereas nitrate ensures
maximum surfactant production in P. aeruginosa [50].
Environmental factors and growth conditions such as pH, temperature, agitation and
aeration are extremely important for the yield and characteristics of the biosurfctant
produced. The pH of the medium plays an important role in the rhamnolipids
production by Pseudomonas sp.: its optimum is at a pH range from 6 to 6.5 and a sharp
decrease is observed above pH 7 [51]. Most biosurfactant productions are reported to be
performed within a temperature range of 25-30°C. In A. paraffineus and Pseudomonas
sp. strain DSM-2874, this temperature range caused alteration in the composition of
biosurfactant production [29].
Aeration and agitation significantly influence the production of biosurfactants, as well
as facilitating the transference of oxygen from the gas phase to the liquid phase.
Margaritis and co-workers [52] when evaluating the influence of aeration on the
biosurfactant biosynthesis by Nocardia erythropolis observed a marked reduction of
biosurfactant yield when agitation speed was increased.
The production of biosurfactant also depends on the concentration of salt, which effects
cellular activity. However, some biosurfactants were not influenced by concentrations
up to 10% (w/v) NaCl, although slight and insignificant reductions in CMC were
detected [29].
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
2.1.4 Towards commercial production of microbial surfactants
Successful commercialization of every biotechnological product depends largely on its
bioprocess economics. Despite their multifarious advantages and diverse potential
applications at present, the prices of microbial surfactants are not competitive with those
of the chemical surfactants, due to their high production costs and low yields [53].
However, different strategies have been adopted to make the process economically
competitive: these include the use of inexpensive raw material; the development of
economical engineering processes, including optimization of culture conditions and the
recovery process; and the development of overproducing strains. Although a large
number of biosurfactant producers have been reported in literature, biosurfactant
research, particularly related to production enhancement and economics, has been
confined mostly to a few genera of microorganisms, such as Bacillus, Pseudomonas and
Candida. A large group of biosurfactant producers belonging to the genera
Rhodococcus, Gordonia, Torulopsis and Acinetobacter have not been adequately
exploited for the economical production of biosurfactants.
One of the primary approaches applied for obtaining increased yields in fermentative
production is the medium optimization. Different elements, such as nitrogen, iron and
manganese, are reported to affect the yield of biosurfactants, for example, the limitation
of nitrogen is reported to enhance biosurfactant production in Pseudomonas aeruginosa
strain BS-2 [54]. Similarly, the addition of iron and manganese to the culture medium
was reported to increase the production of biosurfactant by Bacillus subtilis [55]. The
most effective method used for the optimization of factors is the statistical approach
[56]. There are considerably sources of literature available on medium optimization
using different statistical methods. These include experimental designs such as Plackett-
The choice of raw materials is very important to the overall economics of the process.
Raw materials can make up 10%-30% of the final product cost in most biotechnological
processes [60]. Thus, to reduce this cost it is desirable to use low-cost raw materials.
Some organisms produce biosurfactants only in hydrocarbons, others only in
carbohydrates, and others utilize several substrates, in combination or separately. In the
last few years most biosurfactant production experiments have been aimed at the
development of economical methods for higher yields of biosurfactant by suggesting the
use of low-cost raw materials. Rhaman et al. [61] showed the maximum rhamnolipid 13
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
14
production of 4.31, 2.98, and 1.77 g/L using soybean oil, safflower oil, and glycerol,
respectively. Nitschke and Pastore [62] showed that cassava flour wastewater is an
alternative substrate for surfactant production by Bacillus subtilis and reduces the
surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration
of 3.0 g/L. Attention must be paid, however, to the fact that different carbon sources can
influence the composition of the biosurfactant formed and how it is produced. For
example, Arthrobacter produces 75% extracellular biosurfactant when grown on ethanol
or acetate but with hydrocarbons, it is totally extracellular [63]. The trehalose lipids
produced by Arthrobacter, Nocardia, and Corynebacterium were replaced by sucrose
and fructose lipids when grown on sucrose or fructose [64].
The third approach, using recombinant hyperproducing strains, has still to be adequately
tested, despite the fact that the hyperproducers have been reported to increase yields
several fold. For example, recombinants of Bacillus subtilis MI 113 have been
developed by expressing foreign genes related to surfactin production, resulting in high
production of surfactin on soybean crud residue [65]. Moreover, recombinant strains
often give rise to better product characteristics. For example, Pseudomonas aeruginosa
is an opportunistic pathogen in humans and is, therefore, not suitable as an industrial
strain. To overcome the problem of the pathogenicity of P. aeruginosa, recombinant
Pseudomonas putida and Pseudomonas fluorescens were developed that produced
rhamnolipids by P. aeruginosa considerable amounts [66]. The incorporation of these
hyper-producing strains will boost the industrial biosurfactant production process and
make it possible to commercialize biosurfactants by making the production process
cheaper and safer.
2.1.5 Functional properties of bioemulsifier/biosurfactant
The composition and distribution of the hydrophilic and hydrophobic functional groups
determine the property of an bioemulsifier/biosurfactant. Consequently, it is possible to
select their suitable application in various industrial fields such as, e.g. cosmetics, food,
pharmaceutics, agriculture, mining and oil recovery [29, 67]. Some functional properties
are reported below:
Surface and Interfacial tension is the most important property of surfactant. The
molecules of water are held together by cohesive forces and surfactants reduce surface
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
tension. Surfactin produced by Bacillus sp. is the most effective biosurfactant, reducing
the surface tension of water from 72 mN/m to 27 mN/m [26, 68].
Emulsification is the dispersion degree of one liquid into another leading to the mixing
of two immiscible liquids.
The de-emulsification action breaks up emulsion through the disruption of the stable
surface between the bulk phase and the internal phase. Several industries, such as
mining, food, nuclear fuel reprocessing, cosmetics and pharmaceutical need molecules
able to determine de-emulsification [34].
Wetting is the spreading and penetrating power toward a substance that lowers the
surface tension when added to a liquid.
In the process of foaming, the surfactants become concentrated at a gas-liquid interface
leading to the formation of bubbles through the liquid and on the interface resulting in
foam formation. Surfactin exhibits excellent foaming properties when compared with
SDS [69].
Several biosurfactants/bioemulsifiers are stable at various temperatures, pH and ionic
strength. Bacillus licheniformis produces a biosurfactant, the lichenysis, that is stable up
to 50°C, pH of 4.5-9.0 and at NaCl (50g/L), Ca (25 g/L) concentrations [70]. Similarly,
surfactin from B. subtilis is highly stable at 121°C for 20 min and remains so for 6
months at room temperature at pH range 5-11 and 20 % NaCl [62].
2.1.6 Screening methods for detection of biosurfactant/bioemulsifier
producers
The methods used for a screening of surface-active compounds producing strains are
based on the physical effects of surfactants. Satpute and co-workers [71] reported 11
main methods used to screen, detect or evaluate potential bioemulsifier/biosurfactant
producing microorganisms. These methods can give qualitative and/or quantitative
results, the use of which has advantages and disadvantages. Listed below, are only two
methods that have been used in the present work and that have been considered the most
appropriate for selecting new bacterial strains able to produce
bioemulsifier/biosurfactant.
15
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
16
2.1.6.1 Oil Spreading Assay
The oil spreading assay was developed by Morikawa et al. [25] and is based on the
observation of the putative biosurfactant or sample containing it that is put in contact
with distilled water added with crude oil. If biosurfactant is present, the oil is displaced
and a clearing zone is formed. The diameter of this clearing zone on the oil surface
correlates to surfactant activity, also called oil displacement activity. For pure
biosurfactant a linear correlation between the quantity of surfactant and the clearing
zone diameter is given. It is one of the most effective methods for detecting the presence
of biosurfactant producers.
2.1.6.2 Emulsification index
Another popular assay based on the emulsification capacity of biosurfactants was
developed by Cooper and Goldenberg [26]. Kerosene is added directly to the culture
broth (1:2 v/v), vortexed for 2 minutes and allowed to stand for 24 h. The height of
emulsion is measured by taking the layer formed between the aqueous and kerosene
layers. A number of modifications reported by some authors propose to substitute
kerosene by other pure hydrocarbons (n-hexadecane, iso-octane, cyclohexane, toluene,
xylene). Thus, it is possible to quantify the emulsion index E24 (see below in
“Experimental procedures”) and to asses the emulsification index stability over time in
order to designate the strength of a surfactant.
2.2 EXPERIMENTAL PROCEDURES
2.2.1 Bacterial strains
Bacterial strains used in the present work were previously isolated from different
environments such i.e., polluted sites [72], industrial wastewaters [73], archaeological
sites [74] and abandoned soil [75], or simply obtained from the ENEA-Lilith Strain
Collection of the Microbiology Laboratory of Environmental Characterization,
Prevention, and Recovery Unit of ENEA-Casaccia (Rome). The strains were identified
(rDNA 16S sequencing) and characterized for biotechnology potential in previous
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
works (see Tab.2.2). The rDNA 16S sequences are deposited in GenBank
(http://www.ncbi.nlm.nhi.gov/BLAST).
The eleven bacterial strains were screened to verify their ability to produce biopolymers
with strong superficial and interfacial properties (Surface Active Compounds, SACs).
The strains were selected on the basis of two essential features: they should be non
pathogenic and non spore forming bacteria in order to protect the environment and the
safely of the operators.
Table2.2: List of the tested bacterial strains and their main features.
Strain Phylogenetic affiliation (%similarity of rDNA 16S)
GenBanck Accession Numbera
Source References
TSNRS-4 Ochrobactrum sp. (100) EU249585 Mercareccia Tomb of Tarquinia
Sprocati et al.2008
MCC-A5 Aeromicrobium erythreum (98) JF279932 Soil from an abandoned field located in Piana di Monte Verna (Naples, Italy)
Sprocati et al 2012 MCC-SL5 Duganella nigrescens (99) JF279923
MCC-Z Pedobacter sp.(99) JF279930
MCC-X Gordonia sp.(99) JF279928
MCC-S Massilia sp. (99) JF279920
MCC-E Micromonospora sp. (99) JF279912
MCC-G Nocardia sp. (99) JF279914
MCC-T Porphyrobacter donghaensis (99) JF279925
AGL17 Acinetobacter calcoaceticus (100) EU118781 Abandoned contaminated site of Italsider Bagnoli
Sprocati et al.2006
CONC18 Achromobacter xylosoxidans (99) EU275351 Sludge from a tannery depuration system (Ariston- Naples, Italy)
Tasso et al.2008
a: the GenBank accession number of the tested strains. The strains were identified by 16S rDNA sequence similarity with GenBank data bank (http://www.ncbi.nlm.nhi.gov/BLAST)
2.2.2 Control strains
Two strains of the DSMZ collection (Leibniz –Institut DSMZ-Deutsche Sammlung von
Mikroorganismen und Zelkulturen GmbH) were used as positive controls for
biosurfactant production: Bacillus subtilis (DSMZ 3257) and Pseudomonas aeruginosa
(DSMZ 1128).
Cooper and co-workers [24] and Zhang-Miller [23] reported that the Bacillus subtilis
and Pseudomonas aeruginosa strains produce surfactin and rhamnolipids, respectively
three, and namely Duganella nigrescens MCC-SL5, Massilia sp. MCC-S and
Porphyrobacter donghaensis MCC-T, did not grow on both production media (Tab.
2.4). The highest values obtained every 24h by OST and E24 are shown in Tab.2.3.
Pseudomonas aeruginosa and Bacillus subtilis, used as positive controls, have validated
the OST and emulsification assay, by confirming the data found in literature [76-78]
(see Fig. 2.1). Four bacterial strains, appeared to be of potential interest: in particular
Pedobacter sp. MCC-Z and Gordonia sp. MCC-X, produced SACs on Cooper culture
media, while Acinetobacter calcoaceticus AGL17 and Achromobacter xylosoxidans
CONC18 produced active molecules on both culture media.
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
21
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
21
A B
Fig.2.1:Oil spreading test (A) and emulsification assay (B) of Pseudomonas aeruginosa (1128 DSMZ) in Miller culture media.
Among these strains, Pedobacter sp. MCC-Z is of particular interest because it belongs
to the Sphingobacteriaceae family and the Pedobacter genus [79], a genus not yet
described as a bioemulsifier producer and not yet used in remediation technology.
It showed a higher emulsification activity on the Cooper medium both in whole culture
(E24 68%) and in cell-free supernatant (E24 56%) (see Fig. 2.2) than that obtained from
the type-strain Bacillus subtilis (E24 36%), that was able to produce SACs only on
Cooper’s medium. The presence of activity in cell-free supernatant is important, since it
would allow the use of cell-free product, thus reducing significantly the potential costs
of downstream and purification processes. None of the selected strains showed similar
OST results to those recorded with reference strains (see Tab.2.3).
Fig.2.2:Emulsification assay of bioemulsifier MCCZ strain in Cooper culture media whole cells (A), supernatant free of cells (B).
BA
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
22
Hence, on the basis of the screening results Pedobacter sp., MCC-Z was selected for
further study. This bacteria is a Gram negative and non-flagellated heterotrophic
bacterium; it is a non pathogen and non spore-forming organism, characterized by rod
shaped and pink-colored colonies [79].
Table 2.3: Evaluation of SACs production by selected strains, compared with type-strains, in two different culture media, by emulsification assay (E24 %) and Oil Spreading Test (OST). Data are means of three determinations.
Strain code
Identification (%similarity 16S rDNA)
Zhang-Miller culture media Cooper culture media
E24% a OSTb E24%
a OSTb
Whole culture
Cell-free supernatant
Whole culture
Cell-free supernatant
Whole culture
Cell-free supernatant
Whole culture
Cell-free supernatant
MCC-A5 Aeromicrobium erythreum (98)
35.3 3.7 + + 22.3 17.3 + +
MCC-SL5 Duganella nigrescens (99)
No No
MCC Z Pedobacter sp.(99) No No + + 67.7 56.3 + + MCC-X Gordonia sp.(99) 24.7 4.3 No 52.3 28.7 + +
MCC-S Massilia sp. (99) No No
MCC-E Micromonospora sp.(99)
No No No No 9.0 No No No
MCC-G Nocardia sp.(99) 27.0 No + No 3.7 No No No
MCC-T Porphyrobacter donghaensis (99)
No No
AGL17 Acinetobacter calcoaceticus (100)
47.7 12.0 +++ ++ 42.7 10.3 + +
TSNR 4 Ochrobactrum sp. (100)
No 12.0 16.3 + +
CONC18 Achromobacter xylosoxidans (99)
31.7 48.0 + + 48.3 51.3 + +
Type strain DSMZ 3257
Bacillus subtilis 34.3 4.3 ++++ ++++
Type strain DSMZ 1128
Pseudomonas aeruginosa 43.7 45.0 +++++ +++++
2.3.2 Pre-optimization of culture medium
During the last few years, much research has demonstrated that the composition and
surface properties of the biosurfactant depends not only on the producer strains, but also
on the culture conditions, such as, e.g. the nature of the carbon source, the nitrogen
source and the chemical and physical parameters [46].
Recently, the statistical approach has been applied for the optimization of biosurfactant
production. Kiran et al [80] optimized the production of biosurfactant by
Brevibacterium aureum MSA13 using industrial and agro-industrial solid waste
residues as substrates in solid state culture. Rodrigues et al. [59] optimized medium for
biosurfactants production by Lactobacillus lactis 53 and Streptococcus thermophilus A
CHAPTER 2 – SCREENING OF A BACTERIAL STRAINS
using multifactorial analysis. Jacques et al. [81] optimized cultural conditions for
surfactin production by Bacillu subtilis S499. Franzetti et al. [82] identified the cultural
parameters that influence biosurfactant production by Gordonia sp.BS29 and found the
optimal composition of growth medium for the production.
However, the principal objective of this study was to chemically characterize and
determine the physico-chemical and surface properties of a new bioemulsifier produced
by a microorganism (of which there is no trace in literature) in order to evaluate its
potential application in environmental areas.
For this reason, MSM medium was used in this phase since it has been considered a
suitable medium for the production of biosurfactants from Flavobacteria [27] and for
recovery and purification phase of biosurfactant.
Using the medium it has been possible to assess the effect of glucose concentrations on
biosurfactant production with two reasons in mind: 1) to lower production cost; 2) to
simplify the recovery and purification phases of the bioemulsifier from the culture
medium.
Figure 2.3 shows the time course of extracellular emulsifier production, in terms of
emulsification index E24%, by Pedobacter sp. MCC-Z grown on MSM medium added
of increasing concentrations of glucose, as the only carbon source (0.25, 0.5, 1.0 and 2.0
Figure 2.3: E24% values of cell-free supernatants of Pedobacter sp. MCC-Z cultures in MSM with different glucose concentrations. Samples were taken at 24 h intervals and the values reported are averages of three replicates ±the standard deviation.
23
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
24
The emulsification index E24% was maximum when 0.5% glucose was added to MSM
and reached the peak (64%) at 96 h of inoculation. Furthermore increases of glucose
addition resulted to ineffective in term of both production and productivities.
To characterize the relationship between bacterial growth and SACs production, the
time course of bacterial cell density was monitored. The growth curve and the
emulsifying activity of Pedobacter sp. MCC-Z strain in MSM with 0.5% (w/v) glucose,
as the only carbon source, are shown in Fig. 2.4.
0 24 48 72 96 1200
2
4
6
8
10
0
20
40
60
80
E24%
Incubation time (h)
whole supernatant
logC
FU/m
L
logCFU/mL
Figure 2.4: Time course of bacterial growth and emulsifying activity of bioemulsifiers produced by Pedobacter sp. MCC-Z on whole culture broths and cell-free supernatant. Samples were taken at 24 h intervals. Samples were taken at 24 h intervals and the values reported are averages of three replicates ± the standard deviation.
Results showed that the strain excreted the biopolymers in the medium during the
exponential phase (24-48 h) and its concentration reached the maximum during the late
stationary phase of the growth (96 hours). Interestingly, the emulsifying activity of the
supernatants was slightly lower that observed in the whole culture broth; moreover, the
differences decreased during the fermentation process, and was the lowest at 96 and 120
h incubation.
In physical science the first essential step in the direction of learning any subject is to find principles of numerical reckoning and practicable methods for measuring some quality connected with it. I often say that when you can measure what you are speaking about, and express it in numbers, you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meagre and unsatisfactory kind; it may be the beginning of knowledge, but you have scarcely in your thoughts advanced too the state of Science, whatever the matter may be.
Lord Kelvin
CHAPTER 3 RECOVERY, PURIFICATION AND PHYSICO-CHEMICAL CHARACTERIZATION OF THE
BIOEMULSIFIER PRODUCTION BY PEDOBACTER sp. STRAIN MCC-Z
3.1 INTRODUCTION
3.1.1 Recovery and purification of SACs
The most important step in the production of biotechnological products are the recovery
and purification processes. Generally, these step cost approximately 60% of the total
production amount [71]. Thus, the price of microbial surfactants is quite expensive and
they are not competitive with chemical surfactants. This cost can be reduced through the
use of inexpensive and renewable substrates, by improving product yield and combining
steps [29, 31, 83].
On the other hand, the biosurfactant required for the MEOR (Microbial Enhanced Oil
Recovery) does not necessarily need to be as pure as required in pharmaceutical
preparation, especially in cosmetics and medicine.
The most common SACs recovery methods include extraction, precipitation, and
crystallization. The cells must first be separated and either the cell mass or the
supernatant is extracted for biosurfactants. Settling, flotation, centrifugation, or rotary
vacuum filtration are used for this step. A variety of solvents can be used for product
recovery from the culture broth, such as chloroform-methanol, dichloromethane-
methanol, butanol, ethyl acetate, pentene, hexane, acetic acid and ether. During the
whole of this process the risk of contamination with undesired compounds exist. Further
26
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
27
purification must be carried out by column chromatography, thin layer chromatography,
and/or crystallization.
Several conventional methods known for recovery of SACs are mentioned in Table 3.1.
The choice is dependent on cost and effectiveness.
For example, precipitation of biosurfactant by ammonium sulphate has been reported
for high molecular weight bioemulsifier/biosurfactant, such as Emulsan from
Arthrobacter [6] and extracellular emulsifier from Acinetobacter calcoaceticus [84].
The pellet obtained after centrifugation is dissolved in water and extracted with an equal
volume of hexane for the removal of residues. The product is further purified by a
dialysis procedure, and lyophilized.
Surfactin, glycolipids and lipopeptides can be obtained by acidification of the
supernatants [39]. This method is simple inexpensive and readily available for the
recovery of crude bioemulsifier/biosurfactants. The method of precipitation with ethanol
and acetone, to purify bioemulsifier/biosurfactant has been used by many different
researchers. An example is the isolation of the emulsifier from Pseudomonas,
Acinetobacter and Bacillus. Product recovery yields from the culture broth can be quite
low. Product losses are an important factor to be considered when selecting an
appropriate recovery process. Yields of 30-50% from the recovery steps can effectively
double the cost of the previous steps.
Table 3.1: Downstream processes for recovery/purification of biosurfactants/bioemulsifiers Recovery/purification process Biosurfactant source References
Ammonium sulphate precipitation Arthrobacter RAG 1 Rosenberg et al. 1979
Acinetobacter calcoaceticus Kaplan and Rosenberg 1982 A. venetianus RAG-1 Bach et al.2003 Acid precipitation Bacillus subtilis Lee et al. 2006
Solvent precipitation: • Ethanol Acinetobacter calcoaceticus Phetrong et al. 2008 • Acetone Pseudomonas PG-1 Cameotra et al. 1990 • Methyl tertiary butyl ether Rhodococcus Kuyukina et al. 2001
Ultrafiltration Bacillus subtilis Lin 1997 B. licheniformis Lin 1998 Adsorption and elution on ion exchange chromatography
Pseudomonas sp. Matsufuji, Nakata 1997
Dyalisis and liophilization A. calcoaceticus BD4 Kaplan and Rosenberg 1982Filtration and precipitation P. aeruginosa Turkovskaya, 2001
CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
28
Rhamnolipid from Pseudomonas sp. was purified by ion exchange chromatography
[85]. Ultrafiltration membranes have been used mostly to concentrate and purify
biosurfactant, such as surfactin or rhamnolipids, in one step [86]. This method is fast,
easy and yields highly pure biosurfactant/bioemulsifier. Seamless cellulose tubing
dialysis bags are also used for the purification of bioemulsifier. Kaplan and Rosenberg
[84] reported production of bioemulsifier from A. calcoaceticus BD4, while, Shah and
Prabhune [87] reported a simple method for resolution of sophorolipids using dialysis
tubing. This method allows easy and rapid purification of bioemulsifier at a low cost.
Dialysis and ultrafiltration techniques are commonly used to enhance the purity of
bioemulsifier.
The ability to obtain low and high molecular weight bioemulsifier which is reasonably
pure, requires several extraction and purification steps. These steps are made simpler by
the use of a pure carbon source but, the use of these pure products is extremely
expensive. Indeed, recent research has reported the use of low cost sources such as
frying oils and other waste oils as a main carbon source, which would also be an
environmentally-friendly solution for the recycling of these waste products. Therefore,
future prospects should be focused on the production of SACs using inexpensive carbon
substrates. In this way, the microbial surface active compounds would become a
reasonably attractive alternative to commercial surfactants.
3.1.2 Characteristics and properties of SACs
The term “surfactant” covers a wide diversity of surface active compounds, both
synthetic and biological, which concentrate and alter the conditions at interfaces (air-
water, oil-water and solid-liquid). Accumulation of surfactants at interfaces or surfaces
imply the reduction of the surface tension (air-liquid) or interfaces tension (liquid-
liquid). The surface tension is a contractive tendency of the surface of a liquid that
allows it to resist an external force. The net effect is an inward force at its surface that
causes water to behave as if its surface were covered with stretched elastic. Water has a
high surface tension, 72.8 mN/m at 20°C, compared to that of most other liquids. The
physicochemical characteristics that define an effective surfactant are its ability to
reduce the surface tension of water from 72 to 35 mN/m and to enhance the apparent
water solubility of hydrophobic compounds to form water emulsions (interfacial
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
29
tension, IT, of water/hexadecane from 40 to 1 mN/m) [27]. Surfactin from Bacillus
subtilis can reduce the surface tension of water to 25 mN/m and the interfacial tension
of water/hexadecane to <1 mN/m [88]. Rhamnolipids from P. aeruginosa decrease the
surface tension of water to 26 mN/m, and the interfacial tension of water/hexadecane to
<1 mN/m [89].
The most important property of bioemulsifier is the ability to form a stable emulsion for
months and, in some cases, years. Higher molecular weight SACs are in general better
emulsifiers than low molecular weight SACs. For example, the Sophorolipids from T.
bombicola have been shown to reduce surface tension, but are not good emulsifiers.
Instead, Liposan does not reduce surface tension, but has been used successfully to
emulsify edible oils. This property is especially useful for application in the cosmetics
and food industries.
Surfactants are characterized by properties such as critical micelle concentration
(CMC), hydrophilic-lipophilic balance (HLB), chemical structure and charge.
Surfactants exist in aqueous solution, at low concentration, as monomers or single
molecules; at the CMC, the surfactant molecules begin to spontaneously associate into
structured aggregates such as micelles and vesicles and a drastic change occurs in many
physico-chemical properties, such as surface tension, turbidity or conductivity [90,91].
These aggregates are capable of dissolving hydrophobic contaminants in their
hydrophobic core. The net effect is an increase of apparent aqueous solubility of the
hydrophobic compounds [92, 93]. The CMC depends on surfactant structure,
composition, temperature, ionic strength and the presence of organic additives in the
solutions [94].
Another important parameter is the HLB number of surfactant, it is specific for each
surfactant and indicates the types of oil that it can emulsify. This number is determined
by the relationship of the hydrophilic and the hydrophobic parts of the surfactant
molecule [95]. Surfactants with a low HLB are lipophilic whereas a high HLB is
indicative of better water solubility [96].
Microbial surfactants are not generally affected by environmental conditions such as
temperature, pH and ionic strength. McInerney et al.[70] reported that lichenysin from
CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
30
B. licheniformis JF-2 was not affected by temperature up to 50°C, pH 4.5-9.0 and ionic
strength of NaCl up to 50 g/L.
3.2 EXPERIMENTAL PROCEDURES
3.2.1 Recovery of the extracellular crude bioemulsifier
The culture supernatant containing the crude bioemulsifier was separated from the cells
by centrifugation at 9,000 x g at 25°C for 35 min. The supernatant was filtered through
a 0.45-µm Millipore membrane (Milford, MA, USA), and the filtrate was dialyzed
using 12 kDa cut off dialysis membrane (Sigma-Aldrich, Steinheim, Germany) in order
to separate molecules of less than 12 kDa. The dialyzed solution was concentrated by
evaporation and the concentrate was extracted with n-hexane (4:1, v/v) in a separatory
funnel at 25°C. The white emulsion was separated from the water phase and then
washed four times with additional water. Hexane was removed by rotary evaporation at
50°C under reduced pressure and the residue was then freeze-dried and weighed.
3.2.2 Purification of crude bioemulsifier and emulsifying activity
In order to investigate the nature of the compound, the crude extract was purified. For
purification, the crude water-soluble bioemulsifier was applied to a Sephadex GC-25
(Pharmacia) column. The column was pre-equilibrated and eluted with deionized water
with the flow rate maintained at 1.0 mL/min. Fractions having major emulsifying
activity (>60%) were concentrated and lyophilized for further studies.
The emulsification index (E24%) was employed to quantify the emulsifying activity and
the standard emulsification assay was conducted as previously described [2.2.4.2]. All
measurements were mean values from three independent experiments.
3.2.3 Physico-chemical characterization of Pdb-Z
3.2.3.1 Surface tension measurements
Surface tension measurements were performed by the du Nöuy ring method using a 3S
tensiometer (GBX, Romans sur Isère, France) on Pdb-Z solubilized in Ultra pure MilliQ
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
31
water at concentrations ranging from 0 to 5.0 mg/mL. All determinations were
performed in three replicates, in two separate experiments.
3.2.3.2 Emulsifying activity with various hydrophobic substrates
The hydrocarbon substrate specificity of Pdb-Z was determined by the emulsification
assay as described in Cooper and Goldenberg [26]. However, the standard hydrocarbon
substrate, n-hexane, was substituted by other pure hydrocarbons (n-hexadecane, iso-
octane, cyclohexane, toluene, xylene) or diesel fuel.
The emulsification assay was performed on Pdb-Z samples diluted in distilled water at
different concentrations (0.25-0.5-0.75-1.0 mg/mL). The synthetic surfactants Tween-
20, Tween-80 and Triton X-100 (Sigma Aldrich) at 0.75% w/v were used as reference
compounds. All measurements were mean values from three independent experiments.
3.2.3.3 Stability study: temperature, pH and ionic strength tolerance
Stability studies were conducted to investigate the effect of several physico-chemical
parameters on the emulsifying activity of the bioemulsifier, as described elsewhere [97].
All the tests were carried out on three replicates.
• The effect of the temperature on the bioemulsifier activity was evaluated by
keeping a 1mg/mL solution of Pdb-Z at different temperatures (-80, -20, +7,
+25, +37, +70, +121°C) for 30 min, and then bringing it to room temperature
before the emulsification assays.
• The pH of bioemulsifier solutions was adjusted from 2 to 12 to test the effect of
pH on the emulsification capability using 1 N HCl or 1 N NaOH at a final
bioemulsifier concentration of 1 mg/mL. After 30 minutes, the E24 test for each
pH condition was carried out.
• The effect of the addition of NaCl (5.0-20.0 %, w/v) on emulsion stability was
investigated. After addition of the salt to a 1 mg/mL solution of bioemulsifier
crude extract in deionized water, the emulsifying activity was assessed by the
emulsification index method (E24%) as described earlier.
CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
32
3.3 RESULTS AND DISCUSSION
3.3.1 Purification and emulsifying activity
The dialyzed solution of the crude bioemulsifier was separated by gel filtration
chromatography on Sephadex GC-25. Four fractions separated from the column, named
fraction I - IV, were tested for their emulsifying activity. Fractions I and IV possessed
no emulsifying activity, whereas fractions II and III showed an E24% approximately
equal to 64%. Respective recovery rates for the fractions were found to be 64.5% and
25.7%. Fractions II and III, namely Pdb-Z, were collected, concentrated and
characterized further. An established criterion for emulsion-stabilizing capacity is the
ability of an emulsifier to maintain at least 50% of the original emulsion volume 24 h
after its formation [98]. Therefore our results indicate that Pdb-Z is an efficient
bioemulsifier.
3.3.2 Surface properties of Pdb-Z: Surface tension and CMC value
In order to evaluate the surface properties of Pdb-Z, the surface tension of increasing
Pdb-Z concentrations was determined. Concentrations from 0 to 2 mg/mL reduced the
surface tension from 73.7 ±0.1 to 41.4±0.6 mN/m (n=6) whereas no further decrease
was observed when the concentrations were increased up to 5 mg/mL. A plot of surface
tension versus the log of Pdb-Z concentration is presented in Figure 3.1. The CMC
value for Pdb-Z, calculated as the intersection between two regression lines describing
the curve, was equal to 2.6 mg/mL.
The CMC is the concentration above which Pdb-Z reaches saturation, forming supra-
molecular aggregates. In order to compare Pdb-Z performance with well-characterized
high molecular weight SACs, the surface tension was used to measure the Pdb-Z
effectively whereas the CMC value was used as a measurement of its efficiency as
previously established by Neu [4]. Pdb-Z exhibited comparable ability to reduce the
surface tension with Alasan by A. radioresistens [99] and superior performance when
compared with Emulsan by A. venetianus RAG-1 [7] even though a higher CMC value
was obtained for Pdb-Z in both cases. Nevertheless, Pdb-Z presents CMC values
comparable with Arabic gum (1.7 mg/mL) [100], a commercial emulsifier extensively
used in the food industry, indicating similar efficiency. Recently, Gutiérrez et al. [101]
have characterized the emulsifying properties of a glycoprotein extract produced by a
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CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
Figure 3.3: Emulsifying activity of Pdb-Z (0.75 mg/mL) with several hydrocarbon substrates in comparison with synthetic surfactants (Triton X-100,Tween-20, Tween-80).
3.3.4 Stability study of Pdb-Z: temperature, pH and ionic strength
tolerance
To explore the possibilities of the surfactant applications, a thermal stability analysis of
the crude bioemulsifier was carried out with temperatures between 0°C and 120°C,
revealing that the properties of the surfactants were maintained with the temperature
increase and only a small decrease in emulsifying activity was observed after a thermal
treatment of 80°C (Fig.3.4). The product is thermostable, showing a slight reduction of
E24% with increasing temperatures. Heat stability of surface active compounds has been
reported by Anyanwu et al. [105] (30-100°C), Ilori et al.[102] (20-50°C) and Maneerat
and Phetrong [106] (30-121°C).
CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
36
-100 -80 -60 -40 -20 0 20 40 60 80 100 120 14030
40
50
60
70
E24
%
T (°C)
Heat Stability
Figure 3.4: Heat stability on emulsifying activity.
The effect of pH on biosurfactant activity is shown in Fig.3.5. The bioemulsifier
demonstrated a stable E24 over the range of pH 3-11. Such an effect of pH on surface
active compounds has been reported earlier [105, 106], as have pH sensitive
biosurfactants [102].
0 2 4 6 8 10 12 1425
30
35
40
45
50
55
60
65
70
E24%
pH
pH stability
Figure 3.5: Influence of pH on emulsifying activity.
While the effect of added NaCl (5-20%) on the stability of bioemulsifier from MCC-Z
is shown in Fig.3.6. The addition of NaCl was ineffective in influencing the activity of
bioemulsifiers. Similar results were previously with Pseudomonas nitroreducens
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
37
TSB.MJJ10 bioemulsifier, whereas SDS used as a comparison showed no activity
beyond a 10% NaCl concentration [103]. The tolerance of bioemulsifier to NaCl has
already been shown as being restricted to 5% [103], 9% [106], 12% [105] and 15%
[107] NaCl.
5 10 15 2050
55
60
65
70
E24
%
NaCl (w/v%)
strength salinity
Figure 3.6: Effect of the salt concentration on emulsifying activity.
Emulsion stability in time was verified up to 16 weeks (E24% test, see Fig.3.7). This
performance is comparable to examples of bioemulsifiers produced by strains that can
be found in literature. Lotfabad et al. [108] described a bioemulsifier produced by
Pseudomonas aeruginosa MR01 that showed a maximum emulsifying activity of about
70%, which remained stable for more than 5 months. This extended stability has also
been previously observed for glycoprotein bioemulsifiers produced by a marine
Antarctobacter [97].
CHAPTER 3 – RECOVERY, PURIFICATION AND PHISICO-CHEMICAL CHARACTERIZATION
38
0 5 10 15 2050
55
60
65
70
E24
%
weeks
Time stability
Figure 3.7: Emulsion time stability of Pdb-Z
Wir sehen nur das, was wir wissen.
Johann Wolfgang Goethe
CHAPTER 4 CHEMICAL CHARACTERIZATION OF Pdb-Z
4.1 INTRODUCTION
Microorganisms synthesize an enormous variety of amphipathic molecules that
typically concentrate at the interfaces between hydrophobic and hydrophilic phases or
on gas/liquid or solid/liquid surfaces. As with chemical surfactants, they are able to
reduce surface or interfacial tensions and they have the ability to form molecular
aggregates. These compounds exemplify immense structural/functional diversity and
consequently possess remarkable applications in a wide range of fields. In the last few
years, a growing number of new SAC-producing microorganisms have been described
although their products often remain uncharacterized in terms of their chemical
structures.
Most research on microbial SACs has been confined to few well-characterized
molecules produced by a small number of microbial genera, such as Acinetobacter,
Pseudomonas, Candida, Bacillus, Serratia. Consequently, our understanding of the
diversity, physiological roles and potential applications of microbial SACs is limited to
a relatively narrow spectrum of microbial metabolites and biological systems.
4.1.1 Microbial surfactants and their structures
The most extensively studied biosurfactants are rhamnolipids produced by several
Pseudomonas species. These compound were found to be glycolipids, disaccharides
acylated with long chain fatty acids or hydroxyl fatty acids. Sophorolipids, synthesized
40
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
41
by different species of the yeast Candida (formerly Torulopsis) [110] are composed of
sophorose disaccharide glycosidically linked to a hydroxyl fatty acid.
Trehalolipids contain carbohydrates and long chain aliphatic acid/hydroxyl aliphatic
acids and are the most effective bioemulsifiers produced by Micobacteria,
Corynebacteria and Rhodococcus species [111]. Most of the biosurfactants produced by
rhodococci are trehalose mycolates consisting of a trehalose residue linked by an ester
bond to mycolic acids, long α-alkyl β-hydroxy fatty acids [112].A variety of structurally
different variants is produced by several Bacillus species. Bacillus subtilis produces a
cyclic lipopeptide called surfactin or subtilisin which has been reported as the most
active biosurfactant discovered to date [113]. The most extensively studied
bioemulsifiers are the ones produced by different Acinetobacter species [5]. An example
of a well-characterized high molecular weight SAC is Emulsan, an effective emulsifier
produced by the Acinetobacter lwoffii strain RAG-1 (formerly Acinetobacter
calcoaceticus). Emulsan is a complex polysaccharide that presents a polyphilic structure
being composed of fatty acids attached over the entire molecule to the polysaccharidic
backbone [114, 6]. Different species of Acinetobacter are known to produce protein
polysaccharide complexes. Another well characterized bioemulsifier is Alasan
produced by A. radioresistens KA53, that finds significant application in
bioremediation [99]. Alasan is an alanine containing complex heteropolysaccharide and
protein polymer that stabilizes oil/water emulsions with n-alkanes [8].
Followed by Acinetobacter, Pseudomonas and Bacillus strains, Serratia is one of the
most well-studied bacterium in terms of molecular genetic studies of bioemulsifier
production. Serratia is known to produce extracellular surface active bioemulsifier
[115]. Serratia marcescens produces a cyclic lipopeptide bioemulsifier Serrawettin
which contains 3-hydroxy-C10 fatty acids side chains [116].
Bodour and co-workers reported a new glycolipid class, the flavolipids, produced by a
Flavobacterium strain isolated from soil. Flavolipids exhibit a unique polar moiety
which features citric acid and two cadaverine molecules and display strong surfactant
and emulsifying activities [27].
CHAPTER 4 – CHEMICAL CHARACTERIZATION OF Pdb-Z
4.2 EXPERIMENTAL PROCEDURES
4.2.1 Chemical composition: proteins, lipids and carbohydrates
Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad). After
the addition of 2x Laemmli buffer (SIGMA), the samples were boiled at 100°C for 5
min and resolved using SDS-polyacrylamide gel electrophoresis. Analysis was
performed on the Coomassie blue-stained spots excised from the gels. The spots were
selected for mass spectral identification through the merging of images analysis.
Proteins excised from the gel were reduced, alkylated and digested in situ with trypsin,
as described by Di Luccia et al.[117]. The peptide mixtures were analysed using a CHIP
MS 6520 QTOF equipped with a capillary 1200 HPLC system and a chip cube (Agilent
Technologies). The sample was then fractionated on a C18 reverse-phase capillary
column (75 µmx43mm in the Agilent Technologies chip) at flow rate of 400 nl min-1
with a linear gradient of eluent B (0.1 formic acid in 95% acetonitrile) in A (0.1%
formic acid in 2% acetonitrile) from 7 to 60% in 50 min. Spectra were scanned over the
range of 400-2000 m/z. Analysis software and MSMS spectra were compared with non-
redundant protein databases (NCBInr 20090924, 9760158 sequences) and
UniprotSwissprot (2011, 167910 sequences), with the taxonomy restriction to Homo
sapiens, using MASCOT 2.1 software (Matrix Science, Boston, USA).
The lipid content of Pdb-Z was determined by GC-MS analysis as described by B. Di
Luccia et al.[117]. After an alkaline digestion, the extracted mixture of species was
directly analysed by GC-MS as TMS derivates. For lipid analysis the oven temperature
was increased from 25°C to 90°C in 1 min and held at 90°C for 1 min before increasing
to 140°C at 25°C/min, to 200°C at 5°C/min and finally to 300°C at 10°C/min. Each
species was univocally identified on the basis of retention times and electron impact
fragmentation spectra (NIST library).
Neutral sugars were determined by GC-MS analysis after hydrolysis with methanolic-
HCl at 80°C for 16 h. After neutralization by adding Ag2CO3, the re-N-acetylation was
achieved with 50 µL acetic anhydride and incubating at room temperature overnight.
The trimethylsilylation was carried out in 500 µL SIGMA-SIL-A at 80°C for 20 min.
The sample was dried down under nitrogen, dissolved in 50 µL hexane, and centrifuged
to remove the excess of solid reagents. The hexane supernatant was used for the GC-MS
analysis. GC-MS analyses were performed on a Agilent 7890 GC/5975 MS system 42
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
43
∆
(Agilent technologies) equipped with DB-5MS fused silica capillary column (30 m,
0.25 mm ID, 0.25 µm ft) from J&W.
4.2.2 Fourier Transform Infrared spectroscopy
The main functional groups of the purified bioemulsifier were assigned using Fourier-
transformed infrared (FT-IR) spectroscopy. Pellets for infrared analysis were prepared
by pressing the purified product. FT-IR spectra were recorded covering an area of 600-
4600 cm-1, with 45 accumulated scans and resolution of 8 cm-1,using a IR Affinity-1
with ATR Miracle 10 Shimadzu spectrometer.
4.2.3 1HNMR spectroscopy and Hydrodynamic study of Pdb-Z
The 1H NMR spectra of Pdb-Z were obtained at 600 MHz in D2O and DMSO-d6
solution on a Brucker Avance 600 MHz, equipped with a 5 mm inverse broadband
probe with z-axis gradients. All data were processed with TopSpin software (Brucker). 1H NMR diffusion experiments were performed using the LED sequence with bipolar
gradients [118]. The attenuation measured with this sequence is given by:
6
Equation 4.1
where I/I0 is the normalized signal intensity, D is the diffusion coefficient, δ is the
duration of the gradient pulse, γH is the gyromagnetic ratio of 1H, G is the gradient
strength, Δ is the diffusion time and τ is eddy current delay. Typical acquisition
parameters were: recycle delay time between diffusion experiments 5 s; Δ, 1 s (DMSO)
or 3 s (D2O); δ, 4 ms; τ, 5 ms. For the DMSO sample, hydrodynamic radius (Rh) of an
equivalent spherical particle was calculated using the Stokes-Einstein equation:
Equation 4.1
CHAPTER 4 – CHEMICAL CHARACTERIZATION OF Pdb-Z
Where η is the macroscopic viscosity value of the solvent, T the absolute temperature
and kB the Boltzmann constant. When dealing with the D2O sample, the hydrodynamic
radius was calculated using dioxane as internal standard [119]. The Rh of the particle is
calculated through the following relationship:
44
,
= ( )
Equation 4.2
where Rhdioxane is the hydrodynamic radius of dioxane (0.21 nm) and Ddioxane and Dpart
are the measured diffusion coefficients for dioxane and the particle, respectively.
4.3 RESULTS AND DISCUSSION
4.3.1 Chemical composition of Pdb-Z
The composition of Pdb-Z was 67% of carbohydrates and was composed of galactose,
xylose, N-acetyl glucosamine, galacturonic acid and talose monomer units. In terms of
peak area, galactose was present as major constituent. A similar percentage of galactose
has been described for two surfactants produced by Trichosporon loubieri CLV20 and
Geotrichum sp. CLOA40, which showed a predominance of galactose [120]. However,
although a number of polysaccharides and oligosaccharides from various bacteria and
yeast have been chemically and structurally characterized to reveal the presence of
unusual sugars [121], this is the first study describing a biopolymer from Pedobacter sp.
containing galactose at significantly high levels.
Therefore, Pdb-Z can be considered as similar to galactan polymers. Pdb-Z showed a
lipid content of about 30%, pentadecanoic acid being the major constituent, and 12-
methyl-tridecanoic acid and adipic acid the minor constituents. The weight percentages
of monosaccharides and lipids of Pdb-Z are shown in Table 4.1.
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
45
Table 4.1: Monosaccharide and fatty acid composition of Pdb-Z produced of Pedobacter sp.MCC-Z.
The molecular composition of these biopolymers may have influenced their emulsifying
activity. Kim and co-workers [124, 125] have suggested that both the composition and
the distribution of fatty acids, carbohydrates and proteins in an biosurfactant play an
important role in its emulsifying activity. An example are the bioemulsans produced by
different species of Acinetobacter RAG-1; Emulsan is a complex of an anionic
heteropolysaccharide and protein [126]. Its surface activity is due to the presence of
fatty acids, comprising 15% of the Emulsan dry weight.
Figure 4.1: SDS-PAGE of Pdb-Z
4.3.2 Fourier Transform Infrared and 1HNMR spectroscopy
Pdb-Z was submitted to FT-IR and 1HNMR analyses for identification of the main
functional groups present in the bioemulsifier. In the FT-IR spectrum (Figure 4.2) the
presence of a large broad band at 3300 cm-1 assigned to O-H stretching, was indicative
of significant water and O-H content, typical of polysaccharides. The spectrum also
showed a band at 1654 cm-1 (υ C=O, amide) and another intense band at 1060 cm-1 (υ
C-O-C, ethers). The attribution of the carbonyl band to an amide group was supported
by the presence of bands at 1550 cm-1 (υ N-H, amide). Considering the carbohydrate
structure, a small band at 900 cm-1 can be seen in the spectra. This band is related to
anomers in polysaccharides since the region between 950 and 700 cm-1 is strongly
dependent on the anomeric carbon [127]. Other important absorption bands which can
46
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
47
be seen in FT-IR spectra of Pdb-Z are the ones at 1740 cm-1, assigned to C=O stretching
of acetyl ester bonds, two bands at 2970 and 2880 cm-1, assigned to C-H asymmetric
stretch of CH2 and CH3 groups, respectively [128]. Overall, the FT-IR spectrum
suggested Pdb-Z is predominantly a polysaccharide, although proteins are also present.
Similar infrared spectra were reported for the polysaccharide isolated from Yarrowia
lipolytica IMUFRJ50682 [101] and a water-soluble acidic EPS produced by Gordonia
polyisoprenivorans CCT 7137 [129].
75010001250150017502000250030003500400045001/cm
0
0,075
0,15
0,225
0,3
0,375
0,45
Abs
biosurfactante IR
Figure 4.2: FT-IR spectra of bioemulsifier produced of Pedobacter sp.MCC-Z strain
Proton NMR study in DMSO and D2O (Figures 4.3 and 4.4) also confirmed the
presence of carboxyl, alkyl, methyl and keto groups (5.01 ppm, 4.88 ppm, 4.975 ppm,
ether/ester at 3.477 ppm, alkanes at 1.626 ppm, 1.241 ppm and 0.853 ppm). These
results also supported the previous conclusions of GC/MS analysis of Pdb-Z.
CHAPTER 4 – CHEMICAL CHARACTERIZATION OF Pdb-Z
Figure 4.3:1H spectrum NMR in D2O and of Pdb-Z
Figure 4.4: 1H spectrum NMR in DMSO and of Pdb-Z
4.3.3 Hydrodynamic behaviour
In order to get insights into the dimension of the molecules that are present in the
sample, we conducted a diffusion study using NMR. DMSO is known to break the
inter- and intra-molecular hydrogen bonds of polysaccharides, leading to the dispersion
of aggregates and making it possible to study individual polymer chains. Variable-
gradient 1H-NMR experiments allowed the measurement of the Rh of the different
components of the mixture, through the measurement of D, the diffusion coefficient. In
Table 4.3, the apparent D values for different signals are reported, together with the
48
NEW SACS FROM BACTERIAL STRAINS: PRODUCTION, CHARACTERIZATION AND POTENTIAL APPLICATION IN ENVIRONMENTAL REMEDIATION
49
calculated Rh using equation (4.2). From a hydrodynamic point of view, the mixture
appears to be heterogeneous. Rh values smaller than 0.8 nm are typical of small
molecules. In the sugar region we find two values of around 5 nm, whereas larger
values are observed for two signals in the aliphatic region (in the 11-13 nm range).
Table4.3: Self-diffusion coefficients, D, measured for Pdb-Z and the calculated hydrodynamic radius (Rh) for different signals observed in the DMSO spectrum.
δ (ppm) D/10-10 (m2s-1) Rh (nm) 8.25 6.19 0.19
7.75 0.14 8.34
5.18 0.22 5.30
4.80 1.93 0.60
4.70 0.23 5.09
3.89 0.69 1.68
3.60 6.22 0.19
2.00 0.10 10.99
1.84 0.09 13.43
1.64 4.80 0.24
1.22 1.70 0.68
A different situation was observed in D2O. D measurements for all the signals in the
spectrum appeared to be homogeneous. Table 4.4 shows the D measurements for the
bioemulsifier (Dpart) and dioxane (Ddiox) used as internal standard [119] and the
calculated Rh for the bioemulsifier, using equation (4.3).
A second measurement was performed on a sample diluted 1:10 diluted sample, in order
to determine the effect of concentration on particle dimensions.
Table4.4: Self-diffusion coefficients, D, measured at 300K in D2O for the bioemulsifier (Dpart)
No test 2 42.0 36 +++++ +++++ 3 44.0 42 +++++ +++++
Mean 43.3 39.3 S. D. ±1.1 ±3.0
a: emulsifying activity was expressed as the percentage of the total height occupied by the emulsion. b: diameter of OST; “+”: 0- 5 mm,; “++”: 6 -10 mm; “+++”: 11- 20 mm; “++++”: 21 -50 mm,;“+++++”: > 51 mm
c: average of three replicates dNO: none emulsifying activity
92
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5 Emulsifying activity (E24%) values of Pedobacter sp. strain, MCC-Z at different glucose concentrations.
% Glu ose c E24% (24h)a E24% (48h)a E24% (72h)a E24% (96h)a E24% (120h)a E24% (144h)a